1. Field of the Invention
The present invention relates to a reagent, a reagent kit and a method for analyzing platelets. The present invention also relates to a reagent, a reagent kid and a method for analyzing platelets and reticulocytes.
2. Description of the Related Art
Human blood contains various blood cells such as red blood cells, white blood cells and platelets. Among these, platelets are anucleate cells of 2 to 3 μm in diameter. In normal human blood, there are 150,000 to 350,000 platelets per μl.
However, it is known that the number of platelets in blood is decreased upon affection with thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) or with acute leukemia. It is generally judged that blood transfusion is necessary when the number of platelets in blood is lower than 10000 per μl. Accordingly, rapid and accurate measurement of the number of platelets is important in the clinical field.
One of known methods of measuring platelets is a method of utilizing electrical resistance. This method is a method which involves measuring a pulse of electrical impedance upon passage of a platelet-containing sample between 2 electrodes and analyzing it in a histogram. In the method of utilizing electrical resistance, however, there is a problem that sufficient measurement accuracy cannot be achieved depending on a sample.
Consequently, a method of measuring the number of platelets by using a labeled antibody specific for surface antigen of platelet is known (see American Journal of Clinical Pathologists (2001):115, pp. 460-464). It is known that the method described in this literature is a highly sensitive method, but that the method generally requires a longtime until results are obtained because an antigen-antibody reaction is used in measurement. Accordingly, this method is not suitable as a method of measuring platelets in the clinical field requiring, for example, urgent judgment of whether blood transfusion is required or not.
Reticulocytes are also contained in blood. Reticulocytes are young red blood cells just after release of denucleated erythroblastic cells in bone marrow into peripheral blood. Reticulocytes are characterized in that as traces in the cell maturation process, a small amount of RNA and organelles such as ribosome and mitochondria, which are not contained in mature red blood cells, are contained in their cells.
In the filed of clinical examination, classification and counting of reticulocytes is very important for grasping hematopoiesis in bone marrow in a patient. In a healthy subject with normal myelopoiesis, reticulocytes account for 0.5 to 3.0% of all red blood cells. On the other hand, the number of reticulocytes is decreased in an abnormal state of myelopoiesis (for example, in a suppressed state of myelopoiesis) or increased in an accelerated state of myelopoiesis. Specifically, reticulocytes are decreased during aplastic anemia and chemical therapy for malignant tumor or increased in hemolytic anemia etc.
A method of rapidly measuring cells in blood (that is, blood cells such as white blood cells, reticulocytes and red blood cells) by using the principle of flow cytometry is known. As such measurement method, a method of counting and identifying reticulocytes, red blood cells and platelets in a sample of whole blood, as well as a reagent composition for use in the method, is disclosed in U.S. Pat. No. 6,114,173. In the method, a reagent mixture containing a cationic dye (particularly oxazine 750) is mixed with a sample containing reticulocytes. Then, the scattered light and absorption light of the resulting mixture are measured by flow cytometry. By using the measured scattered light and f absorption light as parameters, reticulocytes are counted and distinguished from red blood cells and platelets.
U.S. Pat. No. 4,882,284 discloses a method of discriminating white blood cells from red blood cells and platelets in whole blood not lysed. In the method, a reagent containing a fluorescent dye which absorbs red light is contacted with whole blood not lysed. It is described therein that an oxazine dye used as the fluorescent dye which absorbs red light stains white blood cells, and thus the white blood cells can be distinguished from red blood cells and platelets by measurement using flow cytometry.
U.S. Pat. No. 5,891,731 discloses a reagent for measurement of reticulocytes, which comprises at least one dye specifically staining reticulocytes and a dye specifically staining white blood cells. This patent also describes that an oxazine-based dye can specifically stain white blood cells.
Fragmented red blood cells, lipid and the like may appear in blood. In measurement of platelets, fragmented red blood cells, lipid and the like are similar in size to platelets and are thus known as contaminants to influence the measurement. The influence of these contaminants is significant particularly in measurement of a sample in which the number of platelets is so low that blood transfusion is necessary. However, the above-mentioned literatures do not describe that platelets are measured more accurately by suppressing the influence of such contaminants.
From the foregoing, there is demand for techniques capable of measuring platelets by distinguishing them more clearly from other blood cells and contaminants in blood such as lipid particles.